1097 - the Cytotoxic Effect of Methotrexate Loaded Bone Cement on the Osteosarcoma Cell Lines

Introduction Polymethylmethacrylate bone cement has been frequently used as an adjunctive material in the limb salvage surgery for malignant bone tumors. The purpose of this study is to investigate the feasibility of bone cement as a vehicle of methotrexate for local treatment of osteosarcoma, and the cytotoxic effect of the eluted methotrexate on the osteosarcoma cells. Also we evaluated the concept of local chemotherapy as a type of adjuvant treatment modality in human osteosarcoma. Materials and Methods Methotrexate were mixed with bone cement to make pellets containing 500mg of cement polymer, 0.25ml of cement monomer, and 5mg, 10mg, 15mg, 20mg, and 50mg of methotrexate respectively. The pellets were put in the 6well plate containing 5ml of HBSS and the elution experiment was performed. The HBSS was changed daily and the concentration of the eluted methotrextate had been daily measured for 28 days with spectrophotometry. The amount and the rate of elution, and the elution pattern were evaluated. Appropriate cell numbers for cytotoxic assay were determined using MTT assay for SaOS2 and MG63 osteosarcoma cell lines respectively. The cells were cultured in 24-well plates. The first column consisted of cells only, and in the second column there were cells and cement not containing methotrexates. The four different concentrations of methotrextate loaded cement pellets were added to each well of the remaining four columns. The culture lasted for two weeks. The numbers of viable cells of each well was measured using MTT assay on the first, third, 5th, 7th, and 14th day. The experiments were repeated three times with the same procedures. The statistical analysis was done with Kruskal Wallis test. Results The eluted amounts of methotrexate from the cement pellets were correlated with the amount of initially mixed methotrexate. The eluted amount of methotrexate was greatest during the first day, then the amount decreased rapidly until the end of the first week, reaching the plateau in the third week. The eluted amount of methotrexate from the pellets averaged 6.1% during the first week, and 9.6% during the four weeks. The concentration of methotrexate was 130 to 10,000 times higher than the minimum inhibitory concentration to inhibit DNA synthesis (1x10M) throughout the experimental period. In the experiment of cytotoxic effect, cement had no influence on the growth of both cell lines. In the groups with methotrexate loaded cement, the cytotoxic effect of the fluted methotrexate revealed that the number of viable cells decreased significantly after three days of culture, and the viable cells were hardly seen after one week. Discussion There have been a few reports about the experiments of bone cement and anticancer drugs in some kinds of skeletal tumors (1,2) except human osteosarcoma. We intended this kind of local chemotherapy to maintain high concentration of anticancer drug during the immediate postoperative period until the beginning of adjuvant chemotherapy after limb salvage surgery for osteosarcoma. In the experiment of drug elution, the concentration of the eluted methotrexate has been sufficiently high to inhibit the growth of osteosarcoma cells for four weeks, which is longer than average duration until the adjuvant chemotherapy after surgery. The elution pattern was similar in the elution of antibiotics (1), rapid elution during the first several days, followed by slow persistent elution for several weeks. The methotrexate did not lose the cytotoxic effect despite high temperature about 70• caused by polymerization of cement. In the experiment of cytotoxicity, MTT assay was useful in evaluating the viable cell numbers even with spectrophotometry instead of ELISA reader. In conclusion, the methotrexate eluted from the bone cement has sufficient cytotoxic effect on the osteosarcoma cells in vitro, and the results suggest that a local chemotherapy using a methotrexate loaded cement may be applicable to the management of osteosarcoma after evaluation of its long term effect. Also this kind of approach may be applied in the treatment of metastatic bone tumors. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2